3 RESULTS AND DISCUSSION
In 2021, TZJ2134 was collected in Shandong Province in China and showed positivity for PRRSV-1 by detection using primer L12 (Table S1). Subsequently, a sequence of TZJ2134 (TZJ2134-L12) was obtained by amplification with the detection primer L12, located in the partial Nsp2 gene (nt 1672-nt 2112 of DV) (Figure 1C). Phylogenetic analysis showed that all Chinese PRRSV-1 isolates belonged to subtype 1 and could be divided into four subgroups (Amervac-like, BJEU06-1-like, HKEU16-like, and NMEU09-1-like isolates) (Figure 1). TZJ2134-L12 belonged to DV-like isolates and shared the highest sequence identity (99.54%) with the DV vaccine strain (Table 1).
To obtain the complete genome of TZJ2134, we designed eight pairs of primers for amplification. Unfortunately, probably due to the low viral load, the whole-genome sequence could not be obtained after repeated attempts, and only two overlapping fragments of TZJ2134 were amplified by using the primers Ly-E and Ly-F (Table S2). Subsequently, the resulting sequences of two overlapping fragments were assembled into a contig (named TZJ2134-(A+B)). Further genetic evolution and homology analyses showed that TZJ2134-(A+B) located at nt 7463-nt 11272 (3810 nt in length) in partial Nsp9, complete Nsp10 and partial Nsp11 (Figure 1C) shared 97.1% nucleotide identity with the DV vaccine strain and 97.4% nucleotide identity with the Amervac vaccine strain (Table 1). To establish a genetic relationship between TZJ2134-(A+B) and other PRRSV-1 isolates, we constructed a phylogenetic tree based on 65 PRRSV-1 strains (Table S1). Phylogenetic analysis showed that TZJ2134-(A+B) was intermediate between Amervac-like isolates and DV-like isolates and formed a separate subgroup (DV+Amervac-like isolates) with PRRS-FR-2014-56-11-1, DK-2011-05-23-9 and OLot/91 strains (Figure 1B).
RDP4 and SimPlot (version 3.5.1) were used to test for recombination of TZJ2134-(A+B). The RDP4 analysis results showed that TZJ2134-(A+B) was a recombinant strain from Amervac and DV vaccine strains with a potential crossover event spanning Nsp10. Additionally, the recombination event was further confirmed by SimPlot 3.5.1, which showed that the recombination breakpoint was approximately located in Nsp10 (nt 9423) (Figure 2A). Based on the putative recombination breakpoint (nt 9243), we divided TZJ2134-(A+B) into two fragments, TZJ2134-A (nt 7463-nt 9423) and TZJ2134-B (nt 9423-nt 11272), for phylogenetic and homology analyses. The results revealed that the homology between the two fragments and the corresponding parent viruses showed high similarity (Table 1). TZJ2134-A shared the highest nucleotide identity (99.17%) with the DV vaccine strain (Table 1) and belonged to DV-like isolates (Figure 2B). TZJ2134-B shared the highest nucleotide identity (99.73%) with the Amervac vaccine strain (Table 1) and belonged to Amervac-like isolates (Figure 2C). Both the DV and Amervac vaccine strains were PRRSV-1 MLV strains. To the best of our knowledge, only two reports, from France and Denmark, have described recombination events between two PRRSV-1 MLV strains (Kvisgaard et al., 2020; Renson et al., 2017). One of them, PRRS-FR-2014-56-11-1, was the first recombinant strain derived from the Amervac vaccine strain and the DV vaccine strain described previously, with recombination events occurring at nt 500 to nt 1370, nt 3646 to nt 4272 and nt 4972 to nt 8430 in ORF1, as determined using RDP4 (Renson et al., 2017). Homology analysis showed that TZJ2134-(A+B) has the highest nucleotide identity (97.6%) with PRRS-FR-2014-56-11-1. PRRS-FR-2014-56-11-1 and TZJ2134-(A+B) are intermediates between Amervac-like isolates and DV-like isolates with DK-2011-05-23-9 and OLot/91 strains in the phylogenetic tree (Figure 1B). The recombinant and phylogenetic analysis results showed that all three viruses were recombinant strains derived from the Amervac vaccine strain and DV vaccine strain but with different recombinant patterns (Figure S1) and formed a novel subgroup (DV+Amervac-like isolates) in the phylogenetic tree (Figure 1B).
In the late 1990s, PRRSV-1 MLVs were usually used to control PRRSV-1 infection in Europe (Chae, 2021). PRRSV-1 MLVs used worldwide include Porcilis PRRS (MSD), Amervac PRRS (Laboratories Hipra S.A.), ReproCyc PRRS EU (Boehringer Ingelheim), Ingelvac PRRSFLEX EU (Boehringer Ingelheim), Pyrsvac-183 (SYVA Laboratories), and Ingelvac PRRSFLEX® EU (Boehringer Ingelheim) (Nan et al., 2017). The Amervac vaccine strain is usually produced from the Amervac PRRS vaccine introduced by Hipra, and the DV vaccine strain is usually produced from the Porcilis® PRRS vaccine introduced by MSD. Both vaccines are often used in western Europe. The present findings confirm that animals infected with the recombinant strain showed a viremia level 10- to 100-fold higher in than that in animals infected with the Amervac or DV vaccine strain, in both inoculated and contact pigs (Eclercy et al., 2019). However, TZJ2134 may have had a low viral load, so the complete genome sequence could not be obtained. This study provides the first genetic evidence of the recombination of the Amervac vaccine strain with the DV vaccine strain in China. As the largest pork importer in the world, in China, the pig industry is vulnerable to the influence of the foreign pig industry (Brockmeier et al., 2012; van Geelen et al., 2018; H. L. Zhang et al., 2018). TZJ2134 may be highly likely to be introduced from Europe via pig trade after recombination abroad, as PRRSV-1 MLV is not currently allowed for use in mainland China. Although recombination of MLV strains is rarely reported, the existence of TZJ2134 is a reminder that surveillance against PRRSV-1 should be strengthened in China.